Stroma-dependent apoptosis in clonal hematopoietic precursors correlates with expression of PYCARD.
The role of the marrow microenvironment in the pathophysiology of myelodysplastic syndromes (MDSs) remains controversial. Using stromal/hematopoietic cell cocultures, we investigated the effects of stroma-derived signals on apoptosis sensitivity in hematopoietic precursors. The leukemia-derived cell line KG1a is resistant to proapoptotic ligands. However, when cocultured with the human stromal cell line HS5 (derived from normal marrow) and exposed to tumor necrosis factor-alpha (TNF-alpha), KG1a cells showed caspase-3 activation and induction of apoptosis. Apoptosis was contact dependent. Identical results were obtained in coculture with primary stroma. Gene-expression profiling of KG1a cells identified coculture-induced up-regulation of various genes involved in apoptosis, including PYCARD. Suppression of PYCARD expression in KG1a by miRNA interfered with apoptosis. Knockdown of the TNF receptor 1 (TNFR1) or TNFR2 in HS5 cells had no effect. However, knockdown of R1 in KG1a cells prevented TNF-alpha-induced apoptosis, while apoptosis was still induced by TNF-alpha-related apoptosis-inducing ligand. Primary CD34(+) cells from MDS marrow, when cocultured with HS5 and TNF-alpha, also underwent apoptosis. In contrast, no apoptosis was observed in CD34(+) cells from the marrow of healthy donors. These data indicate that stroma may convey not only protective effects on hematopoietic cells, but, dependent upon the milieu, may also facilitate apoptosis.
Mhyre
Andrew J
AJ
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Marcondes
A Mario
AM
Spaulding
Emily Y
EY
Deeg
H Joachim
HJ
D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D053468:CARD Signaling Adaptor Proteins; D053148:Caspase 3 / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D002460:Cell Line; D018920:Coculture Techniques; D003598:Cytoskeletal Proteins / Q000096:biosynthesis*; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009190:Myelodysplastic Syndromes / Q000378:metabolism / Q000503:physiopathology; D034622:RNA Interference; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D053468:CARD Signaling Adaptor Proteins; D003598:Cytoskeletal Proteins; C402202:PYCARD protein, human; D018124:Receptors, Tumor Necrosis Factor; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha; D053148:Caspase 3